Effect of South African Herb Hoodia gordonii on Liver Glucolipid Metabolism and PI3K/Akt/FoxO1 Signalling Pathway in db/db Mice / 中国实验方剂学杂志

ObjectiveTo observe the effects of the South African herb Hoodia gordonii （HG） on glucolipid metabolism in diabetic db/db mice and explore the possible mechanisms of HG on the liver of db/db mice based on the phosphoinositide-3 kinase （PI3K）/protein kinase B （Akt）/factor forkhead protein O1 （FoxO1） signaling pathway. MethodA total of 30 db/db mice were randomly divided into five groups according to fasting blood glucose： model group， metformin group （0.195 g·kg-1）， and low dose （0.39 g·kg-1）， medium dose （0.78 g·kg-1）， and high dose （1.56 g·kg-1） HG groups， with six m/m mice in each group， and another six m/m mice were set as normal group. The mice in the normal and model groups were given saline of 9 mL·kg-1 by gavage. Body weight， water intake， and fasting blood glucose of the mice in each group were measured weekly. After six weeks of continuous administration， serum insulin （FINS）， low-density lipoprotein cholesterol （LDL）， total cholesterol （TC）， triglyceride （TG）， alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， urea， and creatinine （CREA） were measured， and liver sections were embedded and stained with hematoxylin-eosin （HE）， periodic acid-Schiff （PAS）， and oil red O. Protein expression of PI3K p85， p-Akt， and p-FoxO1 in liver was detected by immunohistochemistry. The mRNA expression of PI3K， Akt， and FoxO1 in liver tissue was detected by real-time polymerase chain reaction （Real-time PCR）. ResultAfter six weeks of administration intervention， it was found that fasting blood glucose was significantly downregulated in mice in the three HG groups （P<0.05）. The level of islet resistance index was significantly reduced in both the low and medium dose HG groups （P<0.05）. The expression levels of TC， TG， and LDL were reduced in all HG groups （P<0.05， P<0.01）. Pathologically， HG could alleviate hepatocyte steatosis， reduce the volume and content of lipid droplets in liver， and increase the distribution of glycogen granules in liver to some extent in mice. Immunohistochemical assays revealed that PI3K p85 protein expression was significantly increased in the low， medium， and high dose HG groups compared with the model group （P<0.01）. p-Akt protein expression was significantly increased in the medium and high dose HG groups （P<0.05， P<0.01）. p-FoxO1 protein expression was significantly increased in the low， medium， and high dose HG groups （P<0.05， P<0.01）. Compared with the model group， PI3K mRNA was increased in low dose， medium dose， and high dose HG groups （P<0.05）， and Akt mRNA was increased in high dose HG group （P<0.05）. FoxO1 mRNA was decreased in low dose， medium dose， and high dose HG groups （P<0.05）. ConclusionHG can ameliorate the disorder of glucolipid metabolism in db/db mice， which may be related to its activation of the hepatic PI3K/Akt/FoxO1 signaling pathway.

Regulatory Mechanism of Berberine in Inhibiting Apoptosis and Autophagy in Ovarian Granulosa Cells Based on SIRT1/FoxO1 Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the protective effect and regulatory mechanism of berberine （BBR） against the senescence of ovarian granulosa cells. MethodA cell senescence model in the human ovarian granulosa-like tumor （KGN） cell line was induced by H2O2. A control group， a model group， and high-dose （1 μmol·L-1） and low-dose （0.5 μmol·L-1） BBR groups were set up. The cells in the model group and the BBR groups were incubated with 10 μmol·L-1 H2O2 for 40 min. The effect of BBR on KGN cell proliferation was detected by cell counting kit-8 （CCK-8） assay. The effect of BBR on the senescence of KGN cells was detected by β-galactosidase staining. The effects of BBR on the apoptosis and ROS content of KGN cells were detected by flow cytometry. The effects of BBR on the mRNA expression of B-cell lymphoma-2 （Bcl-2）/Bcl-2-associated X protein （Bax）， cysteinyl aspartate-specific protease-3 （Caspase-3）， forkhead transcription factor O1 （FoxO1）， and catalase （CAT） was detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. Western blot was used to detect the effects of BBR on protein expression of silent information regulator1 （SIRT1）， superoxide dismutase 2 （SOD2）， c-Jun N-terminal kinase （JNK）， FoxO1， autophagy-associated protein microtubule-associated protein light chain 3Ⅱ （LC3BⅡ）， mammalian ortholog of yeast Atg6 （Beclin-1）， and ubiquitin-binding protein p62. ResultAfter H2O2 induction for 40 min， the cell proliferation rate of the model group decreased compared with that of the control group （P<0.01）， and the cell proliferation rates of the BBR groups increased compared with that of the model group （P<0.05）. The results of β-galactosidase staining showed that the cells of the model group showed significant senescence compared with those of the control group （P<0.01）， and the cellular senescence in the BBR groups was reduced compared with that of the model group （P<0.01）. As revealed by flow cytometry， compared with the control group， the model group showed increased apoptosis rate （P<0.01）， and compared with the model group， BBR groups showed decreased apoptosis rates （P<0.05）. Meanwhile， the ROS content in the model group increased compared with that in the control group （P<0.01）， and compared with the model group， the BBR groups showed reduced cellular ROS content （P<0.01）. The Real-time PCR results showed that compared with the control group， the model group showed decreased mRNA expression of CAT and Bcl-2/Bax in KGN cells and increased mRNA expression of Caspase-3 and FoxO1 （P<0.05）， and compared with the model group， the BBR groups showed increased mRNA expression of CAT and Bcl-2/Bax （P<0.05） and reduced mRNA expression of Caspase-3 and FoxO1 in KGN cells （P<0.05）. As revealed by Western blot results， SIRT1， SOD2， and p62 protein levels decreased in the model group compared with those in the control group （P<0.01）， and JNK FoxO1， LC3BⅡ， and Beclin-1 protein levels increased （P<0.05）. After BBR intervention， SIRT1， SOD2， and p62 protein levels increased （P<0.01）， and JNK， FoxO1， LC3BⅡ， and Beclin-1 protein levels decreased compared with those in the model group （P<0.05）. ConclusionBBR has an inhibitory effect on ovarian granulosa cell senescence， and the mechanism is related to the inhibition of apoptosis and autophagy mediated by the SIRT1/FoxO1 pathway.

Effect of electroacupuncture on liver Akt/FoxO1 signaling pathway in rats with diabetic fatty / 中国针灸

OBJECTIVE@#To observe the effect of electroacupuncture (EA) on liver protein kinase B (Akt)/forkhead box transcription factor 1 (FoxO1) signaling pathway in Zucker diabetic fatty (ZDF) rats, and to explore the possible mechanism of EA on improving liver insulin resistance of type 2 diabetes mellitus.@*METHODS@#Twelve male 2-month-old ZDF rats were fed with high-fat diet for 4 weeks to establish diabetes model. After modeling, the rats were randomly divided into a model group and an EA group, with 6 rats in each group. In addition, six male Zucker lean (ZL) rats were used as the blank group. The rats in the EA group were treated with EA at bilateral "Zusanli" (ST 36), "Sanyinjiao" (SP 6), "Weiwanxiashu" (EX-B 3), and "Pishu" (BL 20). The ipsilateral "Zusanli" (ST 36) and "Weiwanxiashu" (EX-B 3) were connected to EA device, continuous wave, frequency of 15 Hz, 20 min each time, once a day, six times a week, for a total of 4 weeks. The fasting blood glucose (FBG) in each group was compared before modeling, before intervention and after intervention; the serum levels of insulin (INS) and C-peptide were measured by radioimmunoassay method, and the insulin resistance index (HOMA-IR) was calculated; HE staining method was used to observe the liver tissue morphology; Western blot method was used to detect the protein expression of Akt, FoxO1 and phosphoenolpyruvate carboxykinase (PEPCK) in the liver.@*RESULTS@#Before intervention, compared with the blank group, FBG was increased in the model group and the EA group (P<0.01); after intervention, compared with the model group, FBG in the EA group was decreased (P<0.01). Compared with the blank group, the serum levels of INS and C-peptide, HOMA-IR, and the protein expression of hepatic FoxO1 and PEPCK were increased (P<0.01), while the protein expression of hepatic Akt was decreased (P<0.01) in the model group. Compared with the model group, the serum levels of INS and C-peptide, HOMA-IR, and the protein expression of hepatic FoxO1 and PEPCK were decreased (P<0.01), while the protein expression of hepatic Akt was increased (P<0.01) in the EA group. In the model group, the hepatocytes were structurally disordered and randomly arranged, with a large number of lipid vacuoles in the cytoplasm. In the EA group, the morphology of hepatocytes tended to be normal and lipid vacuoles were decreased.@*CONCLUSION@#EA could reduce FBG and HOMA-IR in ZDF rats, improve liver insulin resistance, which may be related to regulating Akt/FoxO1 signaling pathway.

Effect of acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) on the expression of apoptosis-related factors in rats with premature ovarian insufficiency based on oxidative stress / 中国针灸

OBJECTIVE@#To explore the possible mechanism of acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) on premature ovarian insufficiency (POI) from the perspective of oxidative stress.@*METHODS@#Sixty female SD rats were randomly divided into a blank group, a model group, a sham acupuncture group, a medication group, and an acupuncture group, 12 rats in each group. Except the blank group, the rats in the remaining groups were intraperitoneally injected with cyclophosphamide to establish the POI model. After the model was successfully established, the rats in the acupuncture group were treated with acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28), with a depth of about 12 mm, and the needle was retained for 30 min; the acupuncture was given once a day, for a total of 4 weeks. The rats in the sham acupuncture group were treated with blunt-head needle to tap the skin surface of "Zhibian" (BL 54), without penetrating the skin, once a day for 4 weeks. The rats in the medication group were treated with estradiol valerate by gastric gavage for 4 weeks. After the intervention, the level of reactive oxygen species (ROS) in the ovarian tissue was detected by fluorescence probe; the expression of c-Jun N-terminal kinase (JNK), forkhead box O1 (FoxO1), tumor suppressor gene protein 53 (p53) and p53 up-regulated modulator of apoptosis (Puma) mRNA and protein in ovarian tissue were detected by real-time fluorescence quantitative PCR and Western blot.@*RESULTS@#Compared with the blank group, the level of ROS and the expression of JNK mRNA, p-JNK protein, FoxO1, p53, Puma mRNA and protein in the ovarian tissue in the model group were increased (P<0.01). Compared with the model group, the level of ROS and the expression of p-JNK protein, FoxO1, p53, Puma mRNA and protein in the ovarian tissue in the sham acupuncture group were slightly reduced, but the difference was not statistically significant (P>0.05). The level of ROS and the expression of JNK mRNA, p-JNK protein, FoxO1, p53, Puma mRNA and protein in the ovarian tissue in the acupuncture group and the medication group were reduced (P<0.01).@*CONCLUSION@#Acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) could improve the level of oxidative stress, down-regulate the expression of apoptosis-related factors JNK, FoxO1, p53 and Puma induced by oxidative stress, and inhibit the premature failure of ovarian reserve function caused by apoptosis of ovarian granulosa cells in POI rats.

Effect of Gegen Qinliantang on SIRT1/FoxO1 Autophagy Pathway in db/db Diabetic Mice / 中国实验方剂学杂志

ObjectiveTo validate the alleviating effect of Gegen Qinliantang （GGQLT） on insulin resistance in db/db diabetic mice by regulating the silent information regulator 1 （SIRT1）/forkhead transcription factor O1 （FoxO1） autophagy pathway. MethodSeventy-five SPF-grade spontaneous type 2 diabetic db/db mice and 15 control db/m mice were selected and maintained on regular feed for one week before measuring blood glucose. They were randomly divided into six groups， with 15 mice in each group. The groups included a normal group （physiological saline， 0.2 g·kg-1）， a metformin group （0.2 g·kg-1）， high-， medium-， and low-dose GGQLT groups （31.9， 19.1， 6.9 g·kg-1）， and a model group （physiological saline， 0.2 g·kg-1）. They were orally treated with corresponding drugs for eight weeks， once daily. Fasting blood glucose （FBG） was measured using a Roche glucometer. Serum levels of high-density lipoprotein cholesterol （HDL-C）， low-density lipoprotein cholesterol （LDL-C）， triglyceride （TG）， and total cholesterol （TC） were measured using an automated biochemical analyzer. Fasting serum insulin （INS） levels were determined using enzyme-linked immunosorbent assay （ELISA）， and the homeostasis model assessment of insulin resistance （HOMA-IR） was calculated. Western blot was used to detect the expression of Beclin-1， microtubule-associated protein 1 light chain 3 （LC3）， and SIRT1/FoxO1 autophagy pathway-related proteins in liver tissues. Immunohistochemistry was performed to assess the expression of SIRT1， FoxO1， Beclin-1， and LC3B proteins in liver tissues. Transmission electron microscopy was used to observe the formation of autophagosomes in the liver. ResultCompared with the normal group， the model group showed significant increases in FBG， FINS， HOMA-IR， TC， TG， LDL-C， and HDL-C levels （P<0.01）， and significant increases in the expression of SIRT1， Beclin-1， LC3， and FoxO1 proteins in liver tissues （P<0.01）. Transmission electron microscopy revealed the highest number of autophagosomes in the model group. Compared with the model group， the metformin group and the low-， medium-， and high-dose GGQLT groups showed significant decreases in serum FBG， FINS， HOMA-IR， TC， TG， LDL-C， and HDL-C levels （P<0.05， P<0.01）， significant decreases in the expression of SIRT1， Beclin-1， LC3 （P<0.05， P<0.01）， and up-regulated FoxO1 protein （P<0.01）. Transmission electron microscopy showed a reduction in the degree of autophagy in the treatment groups. Compared with the metformin group， the medium- and high-dose GGQLT groups showed significant decreases in FBG， FINS， and TG levels （P<0.01）， significant decreases in the expression of SIRT1， Beclin-1， and LC3 in liver tissues （P<0.05， P<0.01）， and reduced FoxO1 protein （P<0.01）. The high-dose GGQLT group showed reduced HOMA-IR， TC， LDL-C， and HDL-C levels （P<0.05， P<0.01）. Transmission electron microscopy revealed a significant reduction in autophagosomes in the medium- and high-dose GGQLT groups. ConclusionGGQLT can significantly improve glucose and lipid metabolism disorders， alleviate insulin resistance in db/db mice， and prevent and treat type 2 diabetes by activating the SIRT1/FoxO1 autophagy pathway.

Resveratrol protects against interleukin-1β-induced chondrocyte injury by regulating the silent information regulator 1/frame transcription factor O1 pathway / 中华风湿病学杂志

Objective:To study the effect of resveratrol (RES) on interleukin-1β (IL-1β)-induced chondrocytes and its pathways of action.Methods:Wistar mammary rat chondrocytes were extracted and divided into 5 groups: control group, IL-1β group, RES+IL-1β group, RES+IL-1β+EX-527 [silent information regulator 1 (SIRT1) inhibitor] group and RES+IL-1β+AS [frame transcription factor O1 (FOXO1) inhibitor] group. Quantitative real time polymerase chain reaction (qRT-PCR) was used to detect SIRT1, forkhead FOXO1 and matrix metalloproteinase 3 (MMP-3) mRNA expression. Protein expression of chondrocyte type Ⅱ collagen (Col-Ⅱ) detected by immunofluorescence, and the expression of chondrocyte SIRT1 and p-FOXO1/FOXO1 was measured by Western blot. The expression of chondrocyte inflammatory factors IL-6 and TNF-α was measured by enzyme-linked immunosorbent assay. One-way analysis of variance (ANOVA) was performed and two-way comparisons between groups were made using the least significant difference (LSD) method. P< 0.001 was statistically significant. Results:Compared to normal chondrocytes, the mRNA and protein expressions of Col-Ⅱ, SIRT1, FOXO1 and p-FOXO1/FOXO1 in chondrocytes induced by IL-1β was significantly decreased ( P<0.001). The secretion of tumor necrosis factor (TNF)-α [(24.70±2.84), t=19.24, P<0.001] and IL-6 [(3.35±0.28), t=12.97, P<0.001] was significantly increased, and the mRNA expression of MMP-3 [(2.46± 0.23), t=12.61, P<0.001] was significantly increased. The mRNA and protein expressions of Col-Ⅱ, SIRT1, FOXO1 and p-FOXO1/FOXO1 were significantly increased. The secretion of TNF-α [(12.60±1.05), t=10.14, P<0.001] and IL-6 [(2.00±0.15), t=9.89, P<0.001] was significantly reduced by RES treated IL-1β-induced chondrocytes. mRNA expression of MMP-3 [(1.30±0.14), t=10.460, P<0.001] was decreased. After adding SIRT1 inhibitor EX-527 or FOXO1 inhibitor AS, RES significantly reduced the mRNA and protein expression of Col-Ⅱ, SIRT1, FOXO1 and p-FOXO1/FOXO1 in IL-1β-induced chondrocytes ( P<0.001). The secretion of TNFα and IL-6 was significantly decreased ( P<0.001), and the mRNA expression of MMP-3 was significantly decreased ( P<0.001). Conclusion:RES significantly ameliorates IL-1β-induced cartilage extracellular matrix egradation and inflammatory responses via the SIRT1/FOXO1 pathway.

Effect of Trichosanthis Radix-Polygonati Rhizoma on Glucose and Lipid Metabolism and Hepatic Insulin Resistance in KKAy Mice / 中国实验方剂学杂志

ObjectiveTo observe the effects of the water extracts of Trichosanthis Radix-Polygonati Rhizoma at different ratios on glucose and lipid metabolism in KKAy mice with spontaneous type 2 diabetes and explore the mechanism of the extract in alleviating insulin resistance based on phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）/forkhead box O1 （FoxO1） signaling pathway. MethodThe 8-week-old C57BL/6J male mice were taken as the normal control group， and KKAy male mice of the same age were randomly assigned into a model group， a metformin group， Trichosanthis Radix-Polygonati Rhizoma groups at the ratios of 1∶1 （Trichosanthis Radix 30 g， Polygonati Rhizoma 30 g）， 1∶3 （Trichosanthis Radix 15 g， Polygonati Rhizoma 45 g）， and 1∶5 （Trichosanthis Radix 10 g， Polygonati Rhizoma 50 g） according to blood glucose level and body weight， with 6 mice in each group. The administration lasted for 8 weeks， and the body weight （BW） and fasting blood glucose （FBG） of mice were recorded at the same time points of the 2nd， 4th， 6th， and 8th weeks， respectively. Oral glucose tolerance test （OGTT） was performed at the 7th week. After drug administration， the serum levels of total cholesterol （TC）， triglyceride （TG）， low-density lipoprotein cholesterol （LDL-C）， high-density lipoprotein cholesterol （HDL-C）， and fasting insulin （FINS） were measured， and homeostasis model assessment-insulin resistance （HOMA-IR） index was calculated. The liver tissue samples were stained with hematylin-eosin （HE） and periodic acid-Schiff （PAS） for observation of the fat distribution and glycogen content. The protein levels of PI3K， Akt， p-Akt， FoxO1， and p-FoxO1 in the liver were determined by Western blot. ResultCompared with the normal group， the model group showed increased food intake， FBG， glucose tolerance， FINS， HOMA-IR， TC， TG， and LDL-C （P<0.01）， and down-regulated protein levels of PI3K， Akt， phosphorylaison （p）-Akt， FoxO1， and p-FoxO1 in the liver （P<0.01）. Compared with the model group， Trichosanthis Radix-Polygonati Rhizoma lowered FBG and HOMA-IR （P<0.05， P<0.01）. In particular， the combination at the ratio of 1∶3 showed the best performance （P<0.01） comparable to metformin. Furthermore， Trichosanthis Radix-Polygonati Rhizoma at different ratios lowered blood glucose at different time points of OGTT （P<0.05） and TC and LDL-C （P<0.01）. Additionally， the combination at the ratio of 1∶3 reduced TG （P<0.01）. The liver of mice in the drug administration groups showed regular morphology， with few lipid droplets and rich glycogen. Western blot showed that Trichosanthis Radix-Polygonati Rhizoma up-regulated the protein levels of PI3K and p-Akt， down-regulated the protein level of FoxO1， and up-regulated the protein level of p-FoxO1 （P<0.05）. ConclusionTrichosanthis Radix-Polygonati Rhizoma， especially at the ratio of 1∶3， lowered the FBG， TC， LDL-C， and HOMA-IR index， promoted liver glycogen synthesis， and reduced steatosis in KKAy mice， which may be related to the regulation of PI3K/Akt/FoxO1 signaling pathway in the liver.

Effect of miR-581 on Autophagy of Ovarian Cancer SKOV3 Cells by Regulating FOXO1 / 肿瘤防治研究

Objective To explore the effect and mechanism of miR-581 on the autophagy of ovarian cancer SKOV3 cells. Methods miR-581 mimics and miR-581 NC were transfected into SKOV3 cells, and the transfection efficiency was detected by qRT-PCR. After successful transfection, Western blot was used to detect autophagy-related proteins expression in SKOV3 cells. TargetScanHuman database predicted miR-581 target genes, and Western blot verified the role of miR-581 and target genes. Results Overexpression of miR-581 significantly inhibited the expression of autophagy-related proteins LC3 Ⅱ and Beclin1 (P < 0.01); miR-581 negatively regulated FOXO1 expression (P < 0.01) and FOXO1 promoted autophagy of SKOV3 cells (P < 0.01). Conclusion miR-581 affects the autophagy of ovarian cancer SKOV3 cells by regulating the expression of FOXO1.

Regulatory Effect of Herbal Pair Astragali Radix-Puerariae Lobatae Radix on PI3K/Akt/FoxO1 Signaling Pathway in Rats with T2DM Based on Network Pharmacology / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of herbal pair Astragali Radix-Puerariae Lobatae Radix （AR-PLR） against type 2 diabetes mellitus （T2DM） based on network pharmacology and experimental verification. MethodThe active ingredients and targets of AR and PLR were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP）. The related targets of T2DM were retrieved from disease databases and the common targets of drugs and diseases were extracted. The protein-protein interaction （PPI） network was analyzed and constructed by STRING and the network topology of key targets was analyzed by Cytoscape 3.7.1. Then gene ontology（GO） and Kyoto encyclopedia of genes and genomes（KEGG） enrichment analyses of core targets were carried out by DAVID to explore its possible molecular mechanism. The T2DM model was induced in rats by the high-fat diet combined with tail intravenous injection of streptozocin. The rats were divided into a normal group，a model group，a metformin group，and high-，medium- and low-dose AR-PLR groups. After four weeks of intragastric administration，the serum levels of fasting blood sugar （FBS），fasting insulin（FINS），aspartate aminotransferase（AST），alanine aminotransferase（ALT），triglyceride（TG），total cholesterol（TC），low-density lipoprotein cholesterin（LDL-C），high-density lipoprotein cholesterin （HDL-C），interleukin-6（IL-6）， and tumor necrosis factor-α（TNF-α） of rats in each group were measured. The protein expression of insulin receptor substrate-2（IRS-2），phosphatidylinositol 3-kinase（PI3K），protein kinase B（Akt）， and forkhead box transcription factor O1（FoxO1） in rat liver was detected by Western blot. ResultA total of 131 core targets of AR-PLR in the treatment of T2DM were screened out by network pharmacology， where Akt1，mitogen-activated protein kinase 1 （MAPK1），TNF-α，and IL-6 were critical. As revealed by KEGG enrichment analysis， AR-PLR exerted the hypoglycemic effect mainly through the PI3K/Akt，TNF， and FoxO signaling pathways. Compared with the model group，the high- and medium-dose AR-PLR groups showed reduced FBS and FINS levels and increased glycogen level （P<0.05，P<0.01），all the AR-PLR groups showed decreased levels of AST，ALT，TG， and LDL-C （P<0.05，P<0.01）， the high- and low-dose AR-PLR groups showed decreased TC levels （P<0.05）. Compared with the model group， the high- and medium-dose AR-PLR groups showed reduced levels of IL-6 and TNF-α（P<0.05，P<0.01）， and the high-dose AR-PLR group showed increased expression of IRS-2， Akt， p-Akt， PI3K， and p-PI3K， and decreased expression of FoxO1 protein（P<0.05）. ConclusionAR-PLR has the characteristics of multi-component，Multi-target and multi-pathway in the treatment of T2DM. This herbal pair may regulate the PI3K/Akt/FoxO1 signaling pathway through IL-6， TNF-α， and other targets to affect insulin resistance， glycogen synthesis， gluconeogenesis， glucose transport， inflammation， immune response， and other processes， thereby treating T2DM.

Study on the mechanism of Poria cocos polysaccharides on the regulation of gluconeogenesis in liver of type 2 diabetic mellitus model rats / 中国药房

OBJECTIVE To investigate the effect and mechanism of Poria cocos polysaccharides on the regulation of blood glucose in type 2 diabetes mellitus （T2DM）model rats by phosphatidylinositol 3-kinase（PI3K）/protein kinase B （Akt）/forked box transcription factor O 1（FoxO1）pathway. METHODS SD rats were randomly divided into blank control group （no modeling ，no administration），model group （modeling，no administration ），metformin group （modeling，200 mg/kg）and P. cocos polysaccharide low-dose，medium-dose and high-dose groups （modeling，100，200，400 mg/kg），8 in each group. Except for blank control group ， other groups were given high fat diet combined with streptozotocin to construct the model of T 2DM rats. At the same time ， administration groups were given relevant dose of medicine intragastrically ，and blank control group and model group were given constant volume of water intragastrically ，once a day ，for consecutive 42 days. During the experiment ，general condition and bodyweight of rats were observed every day ；fasting blood glucose （FBG）of rats were collected ，and oral glucose tolerance test were conducted and area under curve （AUC）was calculated the day before last administration. After last medication ，the heart ，liver， kidney organ index were calculated ；the levels of HbA 1c，TC，TG，MDA，SOD，GSH-Px and hepatic glycogen content were detected. HE staining was used to observe the pathological changes of liver and pancreatic tissue ，and the pathological grade score was calculated. Western blot assay was used to detect the protein expressions of p-PI 3K，p-Akt，p-FoxO1， PEPCK and G 6Pase in liver tissues. RESULTS Compared with blank control group ，the rats of model group suffered cc1965@163.com from polydipsia ，polyphagia and polyuria ；the body weight ， the levels of SOD and GSH-Px ，the protein expressions of p-PI 3K，p-Akt and p-FoxO 1 were significantly decreased （P＜0.05）；liver and kidney organ index ，blood glucose level at 0，0.5 and 2 hours after intragastric administration of glucose solution ，AUC， FBG，HbA1c，serum levels of MDA ，TC，TG and hepatic glycogen content ，liver and pancreatic pathological grade score ，the protein expressions of PEPCK and G 6Pase were all increased significantly （P＜0.05）. Compared with model group ，the general condition of rats in P. cocos polysaccharide groups were all improved ，and all of above indicators had been reversed to varying degrees. CONCLUSIONS P. cocos polysaccharide can downregulate protein expressions of PEPCK and G 6Pase which are key enzymes of gluconeogenesis ，inhibit hepatic gluconeogenesis ，effectively decrease blood glucose levels and regulate glucolipid metabolism in T 2DM model rats by weakening oxidative stress and upregulating PI 3K/Akt/FoxO1 pathway.

Dendrocalamus latiflorus and its component rutin exhibit glucose-lowering activities by inhibiting hepatic glucose production via AKT activation

The potential medicinal value of Ma bamboo (Dendrocalamus latiflorus), one of the most popular and economically important bamboo species in China, has been underestimated. In the present study, we found that D. latiflorus leaf extract (DLE) reduced fasting blood glucose levels, body weight, and low-density lipoprotein cholesterol with low liver toxicity in db/db mice. In addition, gene expression profiling was performed and pathway enrichment analysis showed that DLE affected metabolic pathways. Importantly, DLE activated the AKT signaling pathway and reduced glucose production by downregulating glucose-6-phosphatase (G6PC) and phosphoenolpyruvate carboxykinase 1 (PCK1) expression. Moreover, network pharmacology analysis identified rutin as an active component in DLE through targeting insulin growth factor 1 receptor (IGF1R), an upstream signaling transducer of AKT. Due to its hypoglycemic effects and low toxicity, DLE may be considered an adjuvant treatment option for type 2 diabetes patients.

Expression of FOXO1 in Liver Cancer and Its Prognostic Significance: An Analysis Based on TCGA / 肿瘤防治研究

Objective To investigate the expression and prognostic value of FOXO1 gene in liver cancer tissues based on TCGA and HPA databases. Methods The RNA-seq data of FOXO1 gene in liver cancer were downloaded from TCGA. The difference of FOXO1 gene expression between tumor and adjacent tissues was obtained via R software. The correlation between FOXO1 and clinicopathological features of liver cancer patients was analyzed. Survival analysis was carried out to evaluate the prognostic significance of FOXO1 gene expression in liver cancer patients. Univariate and multivariate Cox analyses were performed to explore prognostic factors. The correlation between FOXO1 expression and TIICs in tumor microenvironment was performed by CIBERSORT. KEGG pathways enrichment analysis was performed for the potential function of FOXO1 gene in liver cancer. Results FOXO1 was downregulated in liver cancer tissues compared with normal tissues (P=1.321E-15). Survival analysis showed that high expression of FOXO1 was positively associated with favorable OS of liver cancer patients (P < 0.05). Clinical stage, T and M stages could be prognostic indicators while FOXO1 wasn't an independent prognostic factor in patients with liver cancer. In TME of liver cancer, the expression of FOXO1 was positively correlated with resting memory CD4 T cells and naive B cells while negatively related to activated memory CD4 T cells and macrophages M2. GSEA identified that FOXO1 participated in multiple cancer-related pathways. Conclusion FOXO1 is down-regulated in liver cancer tissues, and its expression level is associated with OS of patients. FOXO1 might be a biomarker related to the prognosis of liver cancer patients and is expected to be a target for diagnosis and treatment of liver cancer.

Expression change of miR-96 in endometrial cancer and its effect on malignant biological behaviors of cancer cells / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the expression changes of miR-96 in endometrial cancer tissues and cells, and to explore its effect on tumor malignant phenotypes as well as the possible mechanisms. Methods:From April 2016 to December 2018, 76 cases of endometrial cancer tissues from 76 patients who were surgically treated in the Department of Obstetrics and Gynecology of our hospital were selected for this study. qPCR was used to detect the expression of miR-96 in human endometrial cancer tissues and cells, and the correlation between the miR-96 expression and the clinicopathological characteristics of patients was analyzed. miR-96 inhibitor was transfected into human endometrial cancer Ishikawa cells in vitro. After transfection, the expression of miR-96 in Ishikawa cells was detected by qPCR; the tumor biological behaviors of Ishikawa cells were detected by CCK-8 test, Clone formation test, Flow cytometry, Scratch test and Transwell test; and the FOXO1 protein expression in Ishikawa cells was detected by WB. At the same time, Dual luciferase reporter gene assay was used to observe the targeting relationship between miR-96 and FOXO1. Results: The results of qPCR showed that the expression of miR-96 was abnormally high in human endometrial cancer cells (JEC, Ishikawa, HEC-1B) and endometrial cancer tissues (all P<0.01), and the expression of miR-96 was closely related to FIGO stage and lymph node metastasis (all P<0.05). After transfection with miR-96 inhibitor, the expression level of miR-96 in Ishikawa cells decreased significantly (P<0.01), the proliferation activity and clone formation ability decreased significantly (all P<0.01), the apoptotic rate increased significantly (P<0.01), and the scratch healing rate and the number of invasive transmembrane cells decreased significantly (P<0.01). Dual luciferase reporter gene assay showed that miR-96 could directly target FOXO1, and WB showed that miR-96 could negatively regulate FOXO1 protein expression in Ishikawa cells (P<0.01). Conclusion: The expression of miR-96 is abnormally high in endometrial cancer tissues and cells. Inhibiting the expression of miR-96 can inhibit the proliferation, invasion and migration of endometrial cancer cells and promote their apoptosis. The mechanism may be related to the targeted regulation of FOXO1.

Ablation of Akt2 and AMPKα2 rescues high fat diet-induced obesity and hepatic steatosis through Parkin-mediated mitophagy

@#Given the opposing effects of Akt and AMP-activated protein kinase (AMPK) on metabolic homeostasis, this study examined the effects of deletion of Akt2 and AMPKα2 on fat diet-induced hepatic steatosis. Akt2–Ampkα2 double knockout (DKO) mice were placed on high fat diet for 5 months. Glucose metabolism, energy homeostasis, cardiac function, lipid accumulation, and hepatic steatosis were examined. DKO mice were lean without anthropometric defects. High fat intake led to adiposity and decreased respiratory exchange ratio (RER) in wild-type (WT) mice, which were ablated in DKO but not Akt2−/− and Ampkα2−/− mice. High fat intake increased blood and hepatic triglycerides and cholesterol, promoted hepatic steatosis and injury in WT mice. These effects were eliminated in DKO but not Akt2−/− and Ampkα2−/− mice. Fat diet promoted fat accumulation, and enlarged adipocyte size, the effect was negated in DKO mice. Fat intake elevated fatty acid synthase (FAS), carbohydrate-responsive element-binding protein (CHREBP), sterol regulatory element-binding protein 1 (SREBP1), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), peroxisome proliferator-activated receptor-α (PPARα), PPARγ, stearoyl-CoA desaturase 1 (SCD-1), phosphoenolpyruvate carboxykinase (PEPCK), glucose 6-phosphatase (G6Pase), and diglyceride O-acyltransferase 1 (DGAT1), the effect was absent in DKO but not Akt2−/− and Ampkα2−/− mice. Fat diet dampened mitophagy, promoted inflammation and phosphorylation of forkhead box protein O1 (FoxO1) and AMPKα1 (Ser485), the effects were eradicated by DKO. Deletion of Parkin effectively nullified DKO-induced metabolic benefits against high fat intake. Liver samples from obese humans displayed lowered microtubule-associated proteins 1A/1B light chain 3B (LC3B), Pink1, Parkin, as well as enhanced phosphorylation of Akt, AMPK (Ser485), and FoxO1, which were consolidated by RNA sequencing (RNAseq) and mass spectrometry analyses from rodent and human livers. These data suggest that concurrent deletion of Akt2 and AMPKα2 offers resilience to fat diet-induced obesity and hepatic steatosis, possibly through preservation of Parkin-mediated mitophagy and lipid metabolism.

Physalin B reduces Aβ secretion through down-regulation of BACE1 expression by activating FoxO1 and inhibiting STAT3 phosphorylation / 中国天然药物

Physalin B (PB), one of the major active steroidal constituents of Solanaceae Physalis plants, has a wide variety of biological activities. We found that PB significantly down-regulated β-amyloid (Aβ) secretion in N2a/APPsw cells. However, the underlying mechanisms are not well understood. In the current study, we investigated the changes in key enzymes involved in β-amyloid precursor protein (APP) metabolism and other APP metabolites by treating N2a/APPsw cells with PB at different concentrations. The results indicated that PB reduced Aβ secretion, which was caused by down-regulation of β-secretase (BACE1) expression, as indicated at both the protein and mRNA levels. Further research revealed that PB regulated BACE1 expression by inducing the activation of forkhead box O1 (FoxO1) and inhibiting the phosphorylation of signal transducer and activator of transcription 3 (STAT3). In addition, the effect of PB on BACE1 expression and Aβ secretion was reversed by treatment with FoxO1 siRNA and STAT3 antagonist S3I-201. In conclusion, these data demonstrated that PB can effectively down-regulate the expression of BACE1 to reduce Aβsecretion by activating the expression of FoxO1 and inhibiting the phosphorylation of STAT3.

Berberine mitigates nonalcoholic hepatic steatosis by downregulating SIRT1-FoxO1-SREBP2 pathway for cholesterol synthesis / 中西医结合学报

OBJECTIVE@#To investigate effects of berberine (BBR) on cholesterol synthesis in HepG2 cells with free fatty acid (FFA)-induced steatosis and to explore the underlying mechanisms.@*METHODS@#A steatosis cell model was induced in HepG2 cell line fed with FFA (0.5 mmol/L, oleic acid:palmitic acid = 2:1), and then treated with three concentrations of BBR; cell viability was assessed with cell counting kit-8 assays. Lipid accumulation in cells was observed through oil red O staining and total cholesterol (TC) content was detected by TC assay. The effects of BBR on cholesterol synthesis mediators were assessed by Western blotting and quantitative polymerase chain reaction. In addition, both silent information regulator 1 (SIRT1) and forkhead box transcription factor O1 (FoxO1) inhibitors were employed for validation.@*RESULTS@#FFA-induced steatosis was successfully established in HepG2 cells. Lipid accumulation and TC content in BBR groups were significantly lower (P < 0.05, P < 0.01), associated with significantly higher mRNA and protein levels of SIRT1(P < 0.05, P < 0.01), significantly lower sterol regulatory element-binding protein 2 (SREBP2) and 3-hydroxy 3-methylglutaryl-CoA reductase levels (P < 0.05, P < 0.01), as well as higher Acetyl-FoxO1 protein level (P < 0.05, P < 0.01) compared to the FFA only group. Both SIRT1 inhibitor SIRT1-IN-1 and FoxO1 inhibitor AS1842856 blocked the BBR-mediated therapeutic effects. Immunofluorescence showed that the increased SIRT1 expression increased FoxO1 deacetylation, and promoted its nuclear translocation.@*CONCLUSION@#BBR can mitigate FFA-induced steatosis in HepG2 cells by activating SIRT1-FoxO1-SREBP2 signal pathway. BBR may emerge as a potential drug candidate for treating nonalcoholic hepatic steatosis.

Effect and mechanism of Angelica dahurica extract on promoting neovascularization maturation of diabetic ulcer / 中草药

Objective: To study the effect and mechanism of Angelica dahurica extract (AD) on neovascularization maturation in db/db mice. Methods: Forty-eight 8-week-old male db/db mice were randomly divided into model group and A. dahurica extract-treated group, and 24 littermate male db/m mice were set as control group. A mouse model of diabetic ulcer was prepared by punching the back. The A. dahuricae extract-treated group was administered with AD 1.8 g/kg ig, and control group and model group were ig administered with the same amount of normal saline for 21 d. The healing condition of mouse wounds was recorded at 2, 4, 7, 11, 14 and 21 d after trauma. The number and maturation of new blood vessels in wound tissues was observed by HE, immunohistochemistry and immunofluorescence staining at 11 d after trauma. Expression of fork head transcription factor (FOXO1) and angiopoietin-2 (Ang-2) in wound tissues were detected by Western blotting and immunohistochemistry. According to different intervention conditions in vitro, endothelial cells were divided into normal glucose and hypoxia group, high glucose and hypoxia group, hypertonic and hypoxic group, high glucose and hypoxia + A. dahurica extract-treated group. Endothelial cells were co-cultured with pericytes under different intervention conditions, and the effects of A. dahurica intervention on endothelial cells' chemotaxis and tubular structure formation in vitro under high glucose and hypoxia were observed. Western blotting was used to detect the levels of FOXO1, p-FOXO1 and Ang-2 proteins and their upstream protein kinase B (Akt) and phosphorylation protein changes in wound tissues and endothelial cells. Results: The wound healing rate of the A. dahurica extract-treated group, the general recovery of the wound, the number of blood vessels and the maturation of new blood vessels in the wound tissue were significantly better than the model group (P < 0.05). FOXO1 and Ang-2 protein expression levels in the A. dahurica extract-treated group were significantly higher than the model group (P < 0.05); p-Akt and p-FOXO1 protein expression in the A. dahurica extract-treated group were lower than model group (P < 0.05). After A. dahurica intervention in endothelial cells under high glucose and hypoxia in vitro, the expression levels of FOXO1 and Ang-2 protein in the cells were significantly reduced (P < 0.05), p-Akt and p-FOXO1 protein expression were significantly increased (P < 0.05); In addition, A. dahurica extract could increase the chemotaxis of endothelial cells to pericytes under high glucose and hypoxia and promote the formation of tubular structures of endothelial cells and pericytes in vitro under high glucose and hypoxia. Conclusion: A. dahurica extract can promote the formation and maturation of neovascularization, so as to improve the rate and quality of wound healing in db/db mice. The mechanism may be related to the down-regulation of FOXO1/Ang-2 pathway, the recruitment of endothelial cells to pericytes, and the promotion of the maturation of wound new blood vessels.

The role of oxidative stress in inhibiting FOXO1 through activating Cdk5 in neuronal damage / 西安交通大学学报(医学版)

Objective: To investigate the role of oxidative stress in inhibiting forkhead box O1 (FOXO1) through activating cyclin-dependent kinase 5 (Cdk5) in neuronal damage. Methods: Cdk5 was coexpressed with p39 in HEK cells together with wild-type FOXO1 or a nonphosphorylatable FOXO1-S249A mutant; FOXO1 phosphorylation was immunoblotted through pS249-FOXO1 antibody which specific recognized FOXO1 Ser249 phosphorylation. Cdk5 or p39 was coexpressed in HEK cells together with FOXO1 and their interaction was explored through Co-IP. In primary cortical culture neurons, various Cdk5 activators (p25, p35, and p39) were overexpressed together with Cdk5. FOXO1 transcriptional activity was tested by luciferase assay. We detected the effect of Cdk5 inhibitor roscovitine on FOXO1 transcriptional activity in primary neurons. We compared FOXO1 subcellular localization of wild type with Cdk5 heterozygous mice cortical brain tissues through cytoplasmic and nuclear extracts. In addition, we administered oxidative stress (H2O2 and glutamate treatment) in primary neurons and examined the effect on the cdk5 activity by Western blot, FOXO1 transcriptional activity by luciferase assay, and neuronal damage through stained caspase3. Results: ① Identification of Cdk5/p39 was capable of phosphorylating the transcription factor FOXO1 at Ser249, and associated with it. ② Activated Cdk5 dramatically inhibited FOXO1 transcriptional activity. In contrast, roscovitine, Cdk5 inhibitor, significantly increased FOXO1 transcriptional activity and led to FOXO1 translocation into the nucleus. ③ FOXO1 protein was found predominately in the nucleus in the Cdk5 heterozygous mice. H2O2 or glutamate treatment promoted p35 to be cleaved to p25, and inhibited FOXO1 transcriptional activity. Importantly, this effect of oxidative stress was abolished when Cdk5 was silenced by shRNA. ⑤ Oxidative stress dramatically increased caspase3 immunostaining in primary neurons. Conclusion: Cdk5/p39 inhibits FOXO1 activity through phosphorylation and association. Moreover, oxidative stress inhibits FOXO1 activity through activating Cdk5, and leads to neuronal death.

Effect of Astragaloside Ⅳ on Regulation of PI3K/Akt/FoxO1 Signal in Kidney of Type 2 Diabetic Nephropathy Rats / 中国实验方剂学杂志

Objective:To study the protective effect of astragaloside (AS) Ⅳ on kidney in type 2 diabetic nephropathy rats and its regulation effect on phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/forkhead box O1(FoxO1) signaling, and investigate the mechanism of glycosides against type 2 diabetic nephropathy. Method:After 6 weeks of high-glucose and high-fat diet, rat models of type 2 diabetes were established by intraperitoneal injection of streptozotocin (STZ) (35 mg·kg-1) and randomly divided into model group, AS-Ⅳ (20, 40, 80 mg·kg-1) groups and metformin hydrochloride (positive) group. After 8 weeks of continuous administration, changes in body weight, kidney index, blood glucose, 24-hour urinary protein, urinary microalbumin, urinary creatinine, serum creatinine, and blood urea nitrogen were measured in each group; hematoxylin-eosin (HE) staining was used to observe the pathological changes of renal tissues; Masson trichrome staining was used to observe the collagen expression level. Periodontium hexaammine silver (PASM) staining was used to observe the changes of basement membrane. Immunohistochemistry assay was used to detect phospho-FoxO1(p-FoxO1) protein expression, and Western blot was used to analyze the expression levels of autophagy marker protein PI3K, microtubule-associated protein 1 light chain 3 (LC3)/Ⅱ, B-cell lymphoma-2 (Bcl-2)/adenovirus E1B19 kDa interacting protein 3 (BNIP3), Beclin1, p-Akt, and Akt. Result:As compared with the normal group, the glomerular basement membrane of the model group was thicker; the extracellular matrix was increased; the mesangial was expanded; the collagen was significantly increased; the PI3K/Akt/FoxO1 signal was increased(PPPPPPConclusion:AS-Ⅳ may increase the autophagic activity of renal cells by inhibiting PI3K/Akt/FoxO1 signal, slowing down the development of type 2 diabetic nephropathy.

Observation on efficacy, pharmacokinetic behaviors and safety of new modified RNA oligonucleotide targeting Foxo-1 via two routes of administration / 药学学报

Foxo-1 plays an important role in development of muscle atrophy, serving as a potential target for therapeutic treatment of the disease. In this study, the Foxo-1 mRNA was targeted by a Foxo-1 specific RNA oligonucleotide modified by 2'-O-methyl and with a butanol tag at the 3'-end. To understand the in vivo significance of new modified RNA oligos, efficacy, pharmacokinetic and safety profiles of the new modified RNA oligonucleotide targeting Foxo-1 were evaluated in mice. All experimental protocols were approved by the Animal Ethics Committee of Institute for Nutrition and Health, Chinese Center for Disease Control and Prevention. The results showed that different doses of the RNA oligonucleotide can reduce the expression of Foxo-1 in mice by two routes of administration, leading to an increase in skeletal muscle mass of the mice. The results of pharmacokinetic evaluation showed that the plasma disappearance curve for the RNA oligonucleotide could be described by a two-compartmental model. The results of safety evaluation showed that no obvious adverse effects on renal and hepatic functions, nor on hematological parameters by intravenous or oral administration of the RNA oligo with a maximum dose of 30 mg·kg-1. Histopathology also did not reveal any significant changes in the morphology of the organs studied. In conclusion, the new modified RNA oligo is safe and effective in mice, providing experimental evidence supporting the significance for its clinical application.